Method for forming inorganic coatings

ABSTRACT

The present invention is directed to a method for forming an inorganic coating on a protein template. The method comprises contacting the template with an anionic polymer interface followed by an inorganic material for a sufficient period of time to allow mineralization of the inorganic material thus forming an inorganic coating on the template. Preferably, the coating is aligned.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application that claims benefit under35 U.S.C. §120 of U.S. patent application Ser. No. 11/289,039, filedNov. 29, 2005, now U.S. Pat. No. 7,727,575 B2, which is a continuationapplication that claims benefit under 35 U.S.C. §120 of InternationalApplication No. PCT/US04/17661, filed Jun. 7, 2004, designating theUnited States, which claims the benefit of priority under 35 U.S.C.119(e) of U.S. Provisional Application No. 60/476,547, filed Jun. 6,2003.

GOVERNMENT SUPPORT

This invention was made with government support under grant DE013405awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present application is directed to a method of forming an inorganiccoating on a protein template.

BACKGROUND OF THE INVENTION

Many biomedical procedures require the provision of healthy tissue tocounteract the disease process or trauma being treated. This work isoften hampered by the tremendous shortage of tissues available fortransplantation and/or grafting. Tissue engineering may ultimatelyprovide alternatives to whole organ or tissue transplantation.

In order to generate engineered tissues, various combinations ofbiomaterials and living cells are currently being investigated. Althoughattention is often focused on the cellular aspects of the engineeringprocess, the design characteristics of the biomaterials also constitutea major challenge in this field.

In recent years, the ability to regenerate tissues and to control theproperties of the regenerated tissue have been investigated by trying tospecifically tune the mechanical or chemical properties of thebiomaterial scaffold (Kim et al., 1997; Kohn et al. 1997). The majorityof this work has involved the incorporation of chemical factors into thematerial during processing, or the tuning of mechanical properties byaltering the constituents of the material.

The foregoing methods have been used in an attempt to utilize chemicalor mechanical signaling to affect changes in the proliferation and/ordifferentiation of cells during tissue regeneration. Despite suchefforts, there remains in the art a need for improved biomaterials,particularly those with a better capacity to support complex tissuegrowth in vitro (in cell culture) and in vivo (upon implantation).

SUMMARY OF THE INVENTION

The present invention provides a method for forming an aligned inorganiccoating on a protein template. Preferably, the coating is aligned. Themethod comprises forming an anionic polymer interface on a proteintemplate and contacting the interface with an inorganic material for asufficient period of time to allow mineralization of the inorganicmaterial, thus forming an inorganic coating on the template. Multiplecycles of mineralization can be performed.

Proteins useful for the protein template include structural proteinssuch as keratins, collagens and silks.

In one preferred embodiment the protein template comprises silk. Thesilk can be processed into various forms, for example, a fiber (e.g.electrospun), a film, a hydrogel, a foam, or a three-dimensional porousmatrix.

In one embodiment, the anionic polymer interface is added to thetemplate during formation of the template, for example, by mixing ananionic polymer with a silk fibroin solution prior to processing thesilk into a fiber, foam, film, hydrogel, or porous matrix.

In another embodiment, the anionic polymer is adsorbed into or onto apreformed protein template.

The anionic polymer interface is preferably polyaspartic acid,polyacrylic acid, or alginic acid. Mixtures may also be used.

In one embodiment, the inorganic material used for mineralization ishydroxyapatite or silica.

In another embodiment, the inorganic material is an inorganic salt, suchas calcium carbonate.

In one preferred embodiment, the template is pre-organized. For example,the template can be pre-organized into aligned fibers or two-dimensionalsurfaces by stretching.

In a further embodiment, a method for forming matrices, tubes, or sheetsof inorganic material is provided. The method comprises (a) forming aprotein template having an anionic polymer interface; contacting thetemplate of step (a) with an inorganic material for a sufficient periodof time to allow mineralization of the inorganic material, thus formingan inorganic coating on the template; and (c) removing the templateafter mineralization of the inorganic material. Multiple cycles ofmineralization can be performed prior to removal of the template.

In another embodiment, therapeutic or biologically active agents areincorporated into the template with an inorganic coating, or areincorporated into the hollow matrices, tubes, or sheets of inorganicmaterial. The therapeutic or biologically active agents can be mixedinto the inorganic coating during formation of the inorganic coating.Alternatively, they can be added to the protein template duringformation of the template, for example, by mixing with a silk fibroinsolution prior to processing the silk into a fiber, foam, film,hydrogel, or porous matrix. Therapeutic or biologically active agentscan also be incorporated into, or coated onto a preformed template.

In another embodiment, a method for forming an aligned inorganic coatingon a protein template is provided that comprises: (a) contacting aprotein template with poly-L-aspartic acid; and (b) contacting thetemplate of step (a) with calcium carbonate for a sufficient period oftime to allow mineralization of the calcium carbonate thus forming aninorganic coating on the template.

Products produced by the methods of the invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scanning electron micrograph of electrospun fiber aftermethanol treatment (no peptides).

FIG. 2 shows CaCO₃ minerals formed on poly(aspartic acid) (poly-Asp)soaking silk fiber.

FIG. 3a shows CaCO₃ minerals formed on control silk membrane (nopeptides). FIG. 3b shows a crystal obtained after removal of silk.

FIG. 4 shows CaCO₃ minerals formed on poly (glutamic acid) soaking silkfiber.

FIG. 5 shows CaCO₃ minerals formed on aspartic acid soaking silk fiber.

FIGS. 6 a and 6 b show fibers with pores after CaCO₃ mineralization onpoly-Asp soaked silk matrix.

FIG. 7 shows TEM images of silk fiber after mineralization.

FIGS. 8 a-d show minerals formed on a poly-Asp incorporated silk fiberwith 10 mg poly-Asp/g silk: (a) before, (b) after removal of silk and200 mg poly-Asp/g silk: (c) before, (d) after removal of silk.

FIGS. 9 a and 9 b show X-ray diffraction pattern of silk matrix before(a) and after mineralization (b).

FIGS. 10 a and 10 b show stretched silk matrix with no poly-Asp (a) andpoly-Asp incorporated (b).

FIGS. 11 a and 11 b show CaCO₃ minerals formed on aligned silk fibers ona poly-Asp soaked matrix (a) and on a poly-Asp incorporated matrix (b).

FIGS. 12 a-d show SEM images of apatite coating on silk fibers withdifferent concentrations of poly-L-aspartic acid: (a) 0, (b) 10, (c) and(d) 200 mg/g silk after one cycle of 10 minute soaking.

FIGS. 13 a-d show SEM images of apatite coating on silk fibers with 200mg poly-Asp/g silk after different soaking periods and cycles: (a) 1minute, 1 cycle, (b) 10 minutes, 3 cycles, (c) 1 hour, 1 cycle, and (d)1 hour, 3 cycles.

FIGS. 14 a-d show an X-ray photoelectron narrow-scan of the C1s and O1sregion of silk electrospun fiber mats. 14 a, C1s 200 mg poly-L-asp/gsilk; 14 b, C1s 0 mg/poly-L-asp/g silk; 14 c, O1s 200 mg poly-L-asp/gsilk; 14 d, O1s 0 mg/poly-L-asp/g silk.

FIGS. 15 a-d show EDS spectra of minerals deposited on fiber mats (200mg poly-L-aspartic acid/g silk) with (a) 10 min, 1 cycle (b) 10 min, 3cycles, (c) 1 hr, 1 cycle, (15 d) 1 hr, 3 cycles.

FIGS. 16 a-d shows X-ray diffraction patterns of silk fibers afterapatite deposition: 16 a shows line (a) no poly-L-asp, 10 minutes, 1cycle and line (b) no poly-L-asp, 10 minutes, 3 cycles; 16 b shows line(c) 200 mg poly-L-asp/g silk, 10 minutes, 1 cycle and line (d) 200 mgpoly-L-asp/g silk, 10 min, 3 cycles; 16 c shows line (e) 200 mgpoly-L-asp/g silk, 1 hour, 1 cycle and line (f) 200 mg poly-L-asp/gsilk, 1 hour, 3 cycles; 16 d shows line (g) hydroxyapatite standard.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for forming an inorganic coatingon a protein template. Preferably, the coating is aligned. The methodcomprises contacting a protein template having an anionic polymerinterface with an inorganic material for a sufficient period of time toallow mineralization of the inorganic material and thus forming aninorganic coating on the template. The interface can be contacted withthe organic material multiple times.

“Aligned”, as described herein, is the regular, controlled or uniformmineralization of the inorganic crystals on the surface of the template.For example, FIG. 2 illustrates an aligned coating while the crystalcoating in FIG. 3 is not aligned.

Proteins useful for the protein template include structural proteinssuch as keratins, collagens and silks.

Preferably, the protein template comprises a silk fibroin. Depending onthe use of the mineralized protein template, the silk fibroin can beprocessed into, for example, silk fibers (e.g., electrospun) silkthreads, silk hydrogels, silk foams, silk meshes, or intothree-dimensional porous silk matrices.

The anionic polymers interact with the protein template to establish asurface that promotes more regular nucleation and growth of inorganiccrystals and include any anionic polymer capable of nucleating themineral used to form the inorganic coating. In the absence of theanionic component only irregular inorganic surfaces are formed, whileregular controlled surfaces form in the presence of this component. Theanionic polymer can be mixed with the template during the processing orformation of the template, e.g. mixed in the silk fibroin solution priorto casting a film or forming a fiber, gel, or three-dimensional porousmatrix. Alternatively, the anionic polymer can be adsorbed orincorporated into or onto a formed template by, for example, soaking thetemplate in an anionic polymer solution. Preferred anionic polymerinterfaces include polyaspartic acid, polyacrylic acid and alginic acid.

Preferably, the amount of anionic polymer to protein can range from 0.1mg/g of protein up to 1:1 or higher.

According to methods of the invention, an inorganic coating is thenformed on the template by contacting the template that has an anionicpolymer interface with an inorganic material for a sufficient timeperiod to allow mineralization of the inorganic coating. The steps formineralization can be performed multiple times.

The inorganic material is preferably an inorganic salt that interactswith the anionic polymer. Most preferred inorganic materials includehydroxyapatite, silica, iron, cadmium, gold salts, and calciumcarbonate.

In a further embodiment, the template is pre-organized to allowformation of aligned inorganic coated fibers or aligned 2D or 3Dsurfaces (for example, see FIGS. 10 and 11 showing aligned inorganiccoatings on a stretched silk matrix and on aligned silk fibersrespectively). The template can be pre-organized by stretching (e.g.stretching of films).

In one preferred embodiment, the template is removed aftermineralization of the inorganic salt leaving hollow tubes or sheets.

As used herein, the term “fibroin” includes silkworm fibroin and insector spider silk protein (Lucas et al., Adv. Protein Chem 13: 107-242(1958)). Preferably, fibroin is obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk protein isobtained, for example, from Bombyx mori, and the spider silk is obtainedfrom Nephila clavipes. In the alternative, the silk proteins suitablefor use in the present invention can be obtained from a solutioncontaining a genetically engineered silk, such as from bacteria, yeast,mammalian cells, transgenic animals or transgenic plants. See, forexample, WO 97/08315 and U.S. Pat. No. 5,245,012.

Methods for generating silk fibers, films, gels, foams, meshes, andthree-dimensional porous matrices are well known in the art. See, e.g.Altman, et al., Biomaterials 24:401, 2003; PCT Publications, WO2004/000915 and WO 2004/001103; and PCT Application No's PCT/US/04/11199and PCT/USA04/00255, which are herein incorporated by reference.

Preferably, a concentrated silk fibroin solution free of organic solventis used. See, e.g. PCT Application number PCT/US/04/11199.

Preferably, before contact with an aqueous solution, the silk fibrointemplate is treated to induce an amorphous β-sheet conformationtransition. Such treatments include, for example, immersion in methanolor stretching. 100% methanol can be used.

Biocompatible polymers can be added to the silk solution to generatecomposite templates in the process of the present invention.Biocompatible polymers useful in the present invention include, forexample, polyethylene oxide (PEO) (U.S. Pat. No. 6,302,848),polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734), collagen (U.S. Pat.No. 6,127,143), fibronectin (U.S. Pat. No. 5,263,992), keratin (U.S.Pat. No. 6,379,690), polyaspartic acid (U.S. Pat. No. 5,015,476),polylysine (U.S. Pat. No. 4,806,355), alginate (U.S. Pat. No.6,372,244), chitosan (U.S. Pat. No. 6,310,188), chitin (U.S. Pat. No.5,093,489), hyaluronic acid (U.S. Pat. No. 387,413), pectin (U.S. Pat.No. 6,325,810), polycaprolactone (U.S. Pat. No. 6,337,198), polylacticacid (U.S. Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No.5,576,881), polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans(U.S. Pat. No. 5,902,800), and polyanhydrides (U.S. Pat. No. 5,270,419).Two or more biocompatible polymers can be used.

In one preferred embodiment, additives such aspharmaceutical/therapeutic agents, or biologically active agents, areincorporated into the template or into the inorganic coating. Forexample, growth factors, pharmaceuticals, or biological components canbe incorporated into the template during the formation of the silk film,fiber, foam, gel, thread, mesh, or three-dimensional matrix.Alternatively, additives can be added during the crystallization processof the inorganic coating, or can be coated onto the template with analigned inorganic coating after its' formation. Additives can also beloaded into or onto the inorganic coating after removal of the template.

The variety of different pharmaceutical/therapeutic agents that can beused in conjunction with the methods of the present invention is vastand includes small molecules, proteins, antibodies, peptides and nucleicacids. In general, therapeutic agents which may be administered via theinvention include, without limitation: anti-infectives such asantibiotics and antiviral agents; chemotherapeutic agents (i.e.anticancer agents); anti-rejection agents; analgesics and analgesiccombinations; anti-inflammatory agents; hormones such as steroids;growth factors (bone morphogenic proteins (i.e. BMP's 1-7), bonemorphogenic-like proteins (i.e. GFD-5, GFD-7 and GFD-8), epidermalgrowth factor (EGF), fibroblast growth factor (i.e. FGF 1-9), plateletderived growth factor (PDGF), insulin like growth factor (IGF-I andIGF-II), transforming growth factors (i.e. TGF-β-III), vascularendothelial growth factor (VEGF)); anti-angiogenic proteins such asendostatin, and other naturally derived or genetically engineeredproteins, polysaccharides, glycoproteins, or lipoproteins. Growthfactors are described in The Cellular and Molecular Basis of BoneFormation and Repair by Vicki Rosen and R. Scott Thies, published by R.G. Landes Company, hereby incorporated herein by reference.Additionally, the templates of the present invention can be used todeliver any type of molecular compound, such as, pharmacologicalmaterials, vitamins, sedatives, steroids, hypnotics, antibiotics,chemotherapeutic agents, prostaglandins, and radiopharmaceuticals. Thepresent invention is suitable for delivery the above materials andothers including but not limited to proteins, peptides, nucleotides,carbohydrates, simple sugars, cells, genes, anti-thrombotics,anti-metabolics, growth factor inhibitor, growth promoters,anticoagulants, antimitotics, fibrinolytics, anti-inflammatory steroids,and monoclonal antibodies.

The therapeutics/pharmaceuticals of the invention may be formulated bymixing with a pharmaceutically acceptable carrier. Any pharmaceuticalcarrier can be used that does not dissolve the template and/or inorganiccoating. The therapeutic agents, may be present as a liquid, a finelydivided solid, or any other appropriate physical form.

Examples of other biologically active agents suitable for use in themethods of the invention include, but are not limited to: cellattachment mediators, such as collagen, elastin, fibronectin,vitronectin, laminin, proteoglycans, or peptides containing knownintegrin binding domains e.g. “RGD” integrin binding sequence, orvariations thereof, that are known to affect cellular attachment(Schaffner P & Dard 2003 Cell Mol Life Sci. January; 60(1):119-32;Hersel U. et al. 2003 Biomaterials November; 24(24):4385-415);biologically active ligands; and substances that enhance or excludeparticular varieties of cellular or tissue ingrowth. Such additives areparticularly useful, in tissue engineering applications. For example,the steps of cellular repopulation of a 3-dimensional scaffold matrixpreferably are conducted in the presence of growth factors effective topromote proliferation of the cultured cells employed to repopulate thematrix. Agents that promote proliferation will be dependent on the celltype employed. For example, when fibroblast cells are employed, a growthfactor for use herein may be fibroblast growth factor (FGF), mostpreferably basic fibroblast growth factor (bFGF) (Human RecombinantbFGF, UPSTATE Biotechnology, Inc.). Other examples of additive agentsthat enhance proliferation or differentiation include, but are notlimited to, osteoinductive substances, such as bone morphogenic proteins(BMP); cytokines, growth factors such as epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-Iand II) TGF-β and the like.

The resultant matrices can then be used to deliver therapeutic agents tocells and tissues. The ability to incorporate, for examplepharmaceutical agents, growth factors and other biological regulators,enzymes or possibly even cells in the construct of the present inventionprovides for stabilization of these components for long term release andstability, as well as better control of activity and release.

In yet another aspect the present invention provides a method forforming an aligned inorganic coating on a template comprising contactingthe template with poly-L-aspartic acid followed by calcium carbonate fora sufficient period of time to allow mineralization of the calciumcarbonate thus forming an inorganic coating on the template.

Finally, there is also provided a product produced by the methodsdescribed herein.

The products produced by these methods offer new options in theformation of scaffolds for biomaterials and tissue engineeringapplications. While the templates are useful in and of themselves, theability to form inorganic coatings with controlled thickness leads tocontrol of mechanical properties (e.g., stiffness) and biologicalinteractions, such as for bone formation. Furthermore, the ability tocontrol these processes allows one to match structural and functionalperformance of scaffolds for specific tissue targets and needs.

As noted above, the underlying template (e.g., protein) can be removedor etched away to generate porous networks, tubes, or lamellar sheets ofinorganic material. These materials are useful directly as biomaterialscaffolds, for control of cell and tissue growth (e.g., as nerveconduits, bone conduits) and for nonbiological applications (e.g.,filtration and separation media, catalysis, decontamination (directly ofif filled with appropriate chemical or enzymes), radar chaff, coatingsin general, and many related needs—for example, inorganic fillers totoughen materials that can also be filled with a second component.

By combining different matrix materials and mediating peptides, themethod of the present invention can be used in the preparation ofaligned silica, hydroxyapatite, and calcium carbonate nanotubes. Theprocess can also be used in the nucleation and growth of inorganicmaterials, i.e. iron, cadmium, and gold salts, among others, withappropriately selected peptides (e.g., histidine-rich, ferratin-like,cysteine-containing).

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Methods for Forming Silk Protein Templates

The templates of the invention, for example the silk fibers, films,foams, gels, and three-dimensional matrices can be formed fromconcentrated silk fibroin solutions that are prepared in the absence oforganic solvent. For example, a silk fibroin solution can be prepared byany conventional method known to one skilled in the art and dialyzedagainst a hygroscopic polymer (e.g. polyethylene glycol (PEG),polyethylene oxide, amylase or sericin) for a sufficient time to resultin an aqueous silk fibroin solution between 10-30% or greater. Theconcentrated silk fibroin solution can then be processed into thedesired template as described in more detail below. Alternatively, meansknown in the art using organic solvents can be used.

Silk film templates can be produced by casting an aqueous silk fibroinsolution. In one embodiment, the film is contacted with water or watervapor, in the absence of alcohol. The film can then be drawn orstretched mono-axially or biaxially. The stretching of a silk blend filminduces molecular alignment of the film and thereby improves themechanical properties of the film. Preferably, the resulting silk blendfilm is from about 60 to about 240 μm thick, however, thicker samplescan easily be formed by using larger volumes or by depositing multiplelayers.

Foams may be made from methods known in the art, including, for example,freeze-drying and gas foaming in which water is the solvent or nitrogenor other gas is the blowing agent, respectively. Alternately the foam ismade by contacting the silk fibroin solution with granular salt. Thepore size of foams can be controlled, for example by adjusting theconcentration of silk fibroin and the particle size of a granular salt(for example, the preferred diameter of the salt particle is betweenabout 50 microns and about 1000 microns). The salts can be monovalent ordivalent. Preferred salts are monovalent, such as NaCl and KCl. Divalentsalts, such as CaCl₂ can also be used. Contacting the concentrated silkfibroin solution with salt is sufficient to induce a conformationalchange of the amorphous silk to a β-sheet structure that is insoluble inthe solution. After formation of the foam, the excess salt is thenextracted, for example, by immersing in water. The resultant porous foamcan then be dried and the foam can be used, for example, as a cellscaffold in biomedical application.

In one embodiment, the foam is a micropatterned foam. Micropatternedfoams can be prepared using, for example, the method set forth in U.S.Pat. No. 6,423,252, the disclosure of which is incorporated herein byreference. The method comprises contacting the concentrated silksolution with a surface of a mold, the mold comprising on at least onesurface thereof a three-dimensional negative configuration of apredetermined micropattern to be disposed on and integral with at leastone surface of the foam, lyophilizing the solution while in contact withthe micropatterned surface of the mold, thereby providing a lyophilized,micropatterned foam, and removing the lyophilized, micropatterned foamfrom the mold. Foams prepared according this method comprise apredetermined and designed micropattern on at least one surface, whichpattern is effective to facilitate tissue repair, ingrowth orregeneration.

Fiber templates may be produced using, for example, wet spinning orelectrospinning. Alternatively, as the concentrated solution has agel-like consistency, a fiber can be pulled directly from the solution.

Electrospinning can be performed by any means known in the art (see, forexample, U.S. Pat. No. 6,110,590). Preferably, a steel capillary tubewith a 1.0 mm internal diameter tip is mounted on an adjustable,electrically insulated stand. Preferably, the capillary tube ismaintained at a high electric potential and mounted in the parallelplate geometry. The capillary tube is preferably connected to a syringefilled with silk solution. Preferably, a constant volume flow rate ismaintained using a syringe pump, set to keep the solution at the tip ofthe tube without dripping. The electric potential, solution flow rate,and the distance between the capillary tip and the collection screen areadjusted so that a stable jet is obtained. Dry or wet fibers arecollected by varying the distance between the capillary tip and thecollection screen.

A collection screen suitable for collecting silk fibers can be a wiremesh, a polymeric mesh, or a water bath. Alternatively and preferably,the collection screen is an aluminum foil. The aluminum foil can becoated with Teflon fluid to make peeling off the silk fibers easier. Oneskilled in the art will be able to readily select other means ofcollecting the fiber solution as it travels through the electric field.The electric potential difference between the capillary tip and thealuminum foil counter electrode is, preferably, gradually increased toabout 12 kV, however, one skilled in the art should be able to adjustthe electric potential to achieve suitable jet stream.

The fiber may also be formed into yarns and fabrics including forexample, woven or weaved fabrics. Alternatively, the fibers may be in anon-woven network.

Silk hydrogel templates can be prepared by methods known in the art. Thesol-gel transition of the concentrated silk fibroin solution can bemodified by changes in silk fibroin concentration, temperature, saltconcentrations (e.g. CaCl₂, NaCl, and KCl), pH, hydrophilic polymers,and the like. Before the sol-gel transition, the concentrated aqueoussilk solution can be placed in a mold or form. The resulting hydrogelcan then be cut into any shape, using, for example a laser.

3-dimensional porous silk matrix templates may be made by (a) gasfoaming, and (b) solvent casting/particulate leaching (Freyman et al.Progress in Materials Science 2001; 46:273-282; Mikos et al. ElectronicJournal of Biotechnology, Accurate, 2000; 3:No. 2; Thomson et al.Biomaterials, 1998; 19:1935-1943; Widmer et al. Biomaterials, 1998;19:1945-1955; Zhang R.& Ma P. Journal of Biomedical Material Science,1999; 44:446-455; Nam et al. Journal of Applied Polymer Science, Vol.81, 3008-30021, (2001); Agrawal et al. Journal of Biomedical Materialresources 2001, 55, 141-150; Harris et al. Journal of BiomedicalMaterial Research 1998, 42, 396-402; Hutmacher D. Journal of biomaterialscience polymer science Edn 2001. 12, 107-124).

One preferred method for material fabrication is leaching. The solventcasting/particulate leaching method involves mixing a water-solubleporogen, for example, NaCl with a viscous silk polymer solution (Freymanet al. Progress in Materials Science 2001; 46:273-282; mikos et al.Electronic Journal of Biotechnology, Accurate, 2000; 3:No. 2; Agrawal etal. Journal of Biomedical Material resources 2001, 55, 141-150;Hutmacher D. Biomaterials 2000. 21, 2529-2543; Hutmacher D. Journal ofbiomaterial science polymer science Edn 2001. 12, 107-124). The mixtureis cast into a Teflon container where the solvent is evaporated. Theresult is a salt/polymer composite. The composite is immersed in waterto leach out the salt, resulting in a porous three-dimensionalstructure.

EXAMPLES Example I Mineralization of CaCO₃ on Poly(asparticacid) SoakedSilk Fiber Materials and Methods

Cocoons of B. mori silkworm silk were kindly supplied by M. Tsukada,Institute of Sericulture, Tsukuba, Japan. PEO with an average molecularweight of 9×10⁵ g/mol (Aldrich) was used in blending. CaCl₂, ammoniumcarbonate, and poly-L-aspartic

acid (sodium salt, MW 11,000) and poly-L-glutamic acid (sodium salt, MW10900) were obtained from Sigma.

Preparation of Electrospun Fiber Matrix

1. Fiber Matrix without Poly-Asp

Silk membranes composed of nanoscale fibers (less than 600 nm indiameter) were fabricated from aqueous silk solutions with PEO byelectrospinning as described earlier^([6]). The obtained membrane wasimmersed into a 90/10(v/v) methanol/water solution for 10 min to inducean amorphous to β-sheet conformational transition of electrospun silkfiber and then washed with water for 24 hours at 37° C. to remove PEO.This process was performed in a shaking incubator and shaking speed was50 rpm. It was expected that just PEO phase would dissolve from the eachfiber. The obtained membrane was used for mineralization.

2. Fiber Matrix with Poly-Asp

Poly-Asp was added into silk/PEO solution before electrospinning. Theobtained membrane was used for mineralization directly without PEOextraction.

Mineralization on Fiber Matrix

Pieces of silk matts (≈0.5 cm²) were placed in 12-well Tissue culturedishes (untreated, well surface area of 3.8 cm²). A calcium chloridesolution (10 mM, 1.5 mL) containing different amounts of poly-Asp(<10mg) was introduced into the wells and incubated for 24 hours at roomtemperature on a rocking table. Following the removal of the peptidesolution, the silk matt was introduced into a fresh well and overlaidwith CaCl₂ (10 mM) solution. The wells were covered with Parafilm, whichwas punctured with a needle.

Crystallization was induced by slow diffusion of ammonium carbonatevapor into 10 mM CaCl₂ in a closed desiccator. (NH₄)₂CO₃ powder wasplaced in four wells of a 12 well tissue culture dish. Slow diffusionwas achieved through needle holes in the Parafilm covering the culturedishes. The experiments were run at 18±1° C.

For experiments using peptide-incorporated membrane, the matt containingpoly-Asp was used directly for mineralization. For control experiment,silk matt without peptide treatment was used.

Scanning Electron Microscope (SEM)

Silk fibers and minerals formed were examined using LEO Gemini 982 FieldEmission Gun SEM for morphology analysis. The surfaces of the specimenswere coated with gold before SEM observation for some samples.Deproteinated samples were obtained by treatment with sodiumhypochlorite.

X-ray Diffraction (XRD)

WAXD experiments employing CuKa radiation on silk fibers and mineralsformed were done using Bruker D8 Discover X-ray diffractometer withGADDS multiwire area detector. 40 kV and 20 mA and 0.5 mm collimator wasused.

Transmission Electron Microscope (TEM)

Silk fibers after mineralization were stained with osmium tetroxidevapor until it turned dark yellow. The stained silk fibers were thenembedded in Epon resin and left at 60° C. for 24 hours. Thin TEM sampleslices were made by microtoming and observed with Philips CM-10 TEM.

Results and Discussion

1. Silk Membrane Characterization

The silk matrix with nanoscale fibers was produced throughelectrospinning process. The fibers have a size of 500±100 nm (FIG. 1),which is about 40 times smaller than that of the native silk fibersafter extraction of sericin.

After methanol treatment, the silk fiber matrix became water insolubleand collapsed resulting in a densely packed mat. However, it wasobserved that the fiber mat swells in aqueous solution, which makes ituseful as matrix for mineral growth. The aqueous solution containing themineralization species filled the interfiber space, creating localenvironment for mineralization. In addition, the swollen hydrated fibermat contracted approxiamately to its original dimension when dried inair.

2. Silk-CaCO₃ Composite

Silk is a highly hydrophobic biopolymer, which is incompatible with thecharged and hydrated nature of the calcium carbonate surface. In thisstudy, the silk surface is functionalized with poly-Asp to generate asuitable surface to induce calcium carbonate nucleation. In one case,the silk fiber was soaked in a poly-Asp solution resulting theadsorption of the peptides. In the other case, the poly-Asp acid wasblended in the silk solution before electrospinning process. In bothcases, the silk composite consisting of a silk fiber core and a mineralcoating was obtained.

Case 1: Mineralization of CaCO₃ on Poly(aspartic acid) Soaked Silk Fiber

After being soaked in poly-Asp for 24 hours at room temperature, thesilk membrane was used for CaCO₃ mineralization. After 2 days ofcrystallization at 18±1° C., the fibers were coated with a uniform layerof CaCO₃ crystals at some areas of the membrane (FIG. 2). As contrast,crystals formed on the membrane without poly-Asp soaking were bigcrystals protruding from the membrane (FIG. 3). The deproteinated sampleshowed that the big crystals are composed of two parts. One part of thecrystals were embedded in the silk membrane with tubular cavity in theshape of fiber, and the other part is in the form of regularrhombohedral calcite crystal morphology. It is apparent that the crystalnuclei formed inside the membrane cavity locally and the continuousgrowth inside the membrane led to the tubular structure.

With peptide adsorption, the fiber surface became negatively chargedbecause of the carboxylate groups on the side chain of poly-Asp. It isreported that the poly-Asp adopted a β-sheet structure under ourexperimental conditions^([7, 12-16]). Falini et al. use gelatin filmswith entrapped poly-Asp to induce the crystallization of differentpolymorphs of calcium carbonate^([12-16]) and the orientedcrystallization is controlled by the β-sheet structure assumed bypoly-Asp. Poly-Asp favors a α-Helical structure^([17, 18]) and undergoesa conformational change to β-sheet once binding Ca²⁺. This conformationchange might facilitate the anchor of the peptides onto silk surfacethat also adopts β-sheet conformation. The arrangement of the peptidesmight provide a form of interfacial complementarity in which the chargedistribution between anions and cations in planes of the mineral latticeis mimicked by the surface arrangement ions bound to peptides exposed atthe fiber surface. The activation energy of nucleation was then loweredand led to nucleation on the fiber surface. If the silk matrix wassoaked in poly-Asp solution for short period (30 min), big crystalsformed instead of small crystals growing from the fiber surface. Anotherfactor affecting the nucleation of CaCO₃ on fiber surface could be thesurface roughness of the fiber, which is observed on the fiber surfaceafter PEO extraction. The concave features on fiber surface can giverise to a high spatial charge density and three-dimensional clusteringof Ca²⁺ and are good nucleation sites.

The free poly-Asp trapped inside the silk matrix macromoleculescoordinates with the adsorbed peptides to mediate the mineralization. Ifthe silk fiber matrix was washed with CaCl₂ solution after soaking inpoly-Asp solution (in CaCl₂) or the moisture trapped inside the matrixwas removed by filter paper, big CaCO₃ crystals appeared (data notshown). In contrast, if the peptide-soaked silk fiber matrix was useddirectly, the silk fiber was coated with CaCO₃ minerals. To furtherverify the importance of the existence of the free poly-Asp, experimentsby soaking the silk mat in CaCl₂ solution with different amounts ofpeptides were carried out. Coating of the fiber is only obtained at ahigh level of poly-Asp. The anchored peptides act as template for thegrowth of minerals while free poly-Asp can inhibit the growth of theminerals towards the bulk solution, which leads to the coating ofminerals on silk fiber. This template-inhibition mechanism also appliedto the situation of formation of inorganic film on two-dimensionalsubstrates.

The importance of polypeptide conformation on the crystallization ofcalcium carbonate was evaluated by comparing the behavior of poly-Aspwith those of poly (glutamatic acid) (poly-Glu) and monomer of poly-Asp,aspartic acid. Poly-Glu adopts random coil conformation in theconditions where the poly (aspartic acid) adopts β-sheet conformation.FIG. 4 showed how the adsorption of the poly-Glu influences thecrystallization of CaCO₃. No uniform coating of CaCO₃ was obtained. Inthe case of simple aspartic acid, no big difference was observedcompared with the control experiment without peptides (FIG. 5), whichmay be due to the poor adsorption on silk surface and retention insidesilk matrix.

An interesting feature that can be observed from SEM analysis is theexpansion of the fiber during mineralization. Pores were observed onpartially coated fiber (FIG. 6 a) or at the end of the coated fiber(FIG. 6b ). TEM (FIG. 7) images also showed big pores on the crosssection of the coated fiber, while pore size is smaller in the case ofuncoated fiber. The electrospun fiber has porous structure. Thecontinuous crystal growth in the tangential and radial direction aroundthe fiber convex surface caused the fiber expansion leaving bigger poresinside fiber. After the CaCO₃ minerals were dissolved by treatment with1M HCl, SEM analysis showed that the fiber resumed its original size.This indicated the strong interaction between the crystals and the fibersurface and the crystals formed on fiber surface via epitaxial growth.

Case 2: Mineralization of CaCO₃ on Poly (Aspartic Acid) IncorporatedSilk Fiber

A layer of CaCO₃ crystals was also formed on the poly-Asp incorporatedfibers (FIG. 8). Compared to case 1, this is a more efficient method toimprove the attachment of peptides on silk fiber surface. Normally, alarge amount of peptides is needed to reach a high adsorption of thepoly-Asp. The growth of the crystals on peptide incorporated fibersaligned along the fiber axis, which may be due to the orientation of thepoly-Asp and silk molecules. The fiber showed some birefringence underpolarized microscope before methanol treatment. It is generally assumedthat in silk, birefringence is positively correlated with molecularorientation and density, forming more so called ‘β-sheet crystalareas’^([19]). In the case of electrospun fiber, the birefringence iscaused by the orientation of silk molecule, but not necessarily formingβ-sheet structure, because FTIR does not show adsorbsion peaks forβ-sheet structure. It is possible that poly-Asp also adopted orientationalong fiber axes during the electrospinning. The phase separation ofsilk and PEO leads to parallel silk and PEO bands on fiber surface. Thealigned poly-Asp forms a gel-like structure along the silk fiber andtriggers the nucleation of minerals, while released poly-Asp suppressthe growth. Therefore, the aligned growth of minerals was obtained. Themineralized silk fibers were characterized by X-ray diffraction (XRD)and the inorganic phase observed was calcite (FIG. 9). Since fibers areorientated randomly and the curved feature of the fiber, no specificorientation of inorganic crystals was observed by XRD.

Case 3: Mineralization of CaCO₃ on Stretched (Orientated) Silk Fiber

By stretching the silk fibrous mat, it is possible to orientate thefibers (FIG. 10). In this way, an aligned silk matrix is obtained andused as matrix ordered material structure synthesis. Unlike thenon-stretched fiber, the stretched fiber become partiallywater-insoluble after stretching even without methanol treatment, whichsuggest that the stretching can improve the molecular orientation offiber. After mineralization, an ordered fibrous silk-CaCO₃ compositestructure was obtained (FIG. 11). By removing the silk component, anordered inorganic structure of CaCO₃ fiber can be obtained.

Case 4: Apatite Growth on Electrospinning Silk Fibers

An alternative soaking process was used to deposit apatite on silkfibers^([20, 21]). First, a silk mat of 5×10 mm was soaked in 200 mMCaCl₂ solution (buffered with 50 mM Tris·HCl, pH 7.4 at 37° C.) forpredetermined period of time. The silk mat was then transferred to a 120mM Na₂HPO₄ solution after excessive moisture was removed by blotting onfilter paper and soaked the predetermined period of time. Soaking wasrepeated for a specific number of cycles.

Aligned growth of the apatite along fiber axes was obtained when thepoly-L-aspartic acid concentration was 200 mg/g silk (FIG. 12). Ascomparison, scattered apatite particles were deposited on silk fiberswithout poly-L-aspartic acid and only randomly oriented apatiteparticles formed on silk fibers at a low concentration ofpoly-L-aspartic acid. FIG. 13 showed the effect of soaking period andcycles on the deposition of apatite on silk fibers. EDS (X-rayEnergy-Dispersive Spectroscopy) and XRD analysis of samples after 10minutes/3 cycles soaking showed the minerals formed were hydroxyapatite.A Ca/P ratio of 1.67 was obtained by EDS, which is characteristic ofhydroxyapatite. XRD pattern also revealed that hydroxyapatite formed onsilk fibers.

Example II Silk Apatite Composites from Electrospun Fibers

The objective of the present example was to use electrospun silk fibersas nucleation template for growth of apatite as a route to generate newbiomaterials with potential utility for bone-related applications. Theelectrospinning process offers an alternative approach to protein fiberformation that can generate nanometer diameter fibers, a useful featurein some biomaterial and tissue engineering applications due to theincreased surface area⁴²⁻⁴⁶. Electrospinning has been utilized togenerate nanometer diameter fibers from recombinant elastin proteins⁴⁷,silk-like proteins⁴⁸⁻⁵⁰, silkworm silk fibroin⁵¹, type Icollagen^(45,52,53) and spider dragline silk⁵⁴. If electropsun polymerfibers generate structures suitable for control of functional grouppatterning, then such templates should be effective for apatitenucleation and crystal growth, leading to new protein-based composites.Since silk fibroin self organizes due to hydrophilic/hydrophobictriblock sequences⁵⁵, pattern control is a controllable outcome⁵⁶. Inthe present example, the controlled growth of apatite on electrospunsilk fibroin fibers functionalized with poly-L-aspartic acid wasdemonstrated. Poly-L-aspartic acid was blended in the polymer aqueoussolution prior to electrospinning. The structural and morphologicaldetails of these new composites are described, along with assessments ofthe mechanical properties of these new biomaterial composite matrices.

Materials and Methods

Cocoons of B. mori silkworm silk were kindly supplied by M. Tsukada,Institute of Sericulture, Tsukuba, Japan. PEO with an average molecularweight of 9×10⁵ g/mol (Aldrich) was used in blending. CaCl₂, ammoniumcarbonate, poly-L-aspartic acid (sodium salt, Mw 11,000), andpoly-L-lysine (Mw 22,000) were obtained from Sigma (St. Louis, Mo.).Hydroxyapatite standard was obtained from NIST (National Institute ofStandards and Technology, Gaithersburg, Md.) with a Ca/P of 1.664±0.005.

Preparation of Electrospun Fiber Matrices

Silk membranes composed of nanoscale fibers were fabricated from aqueoussilk solutions with PEO by electrospinning as described earlier⁵¹. Thedistance between the capillary tip and the collector was 21.5 cm and theapplied voltage was 12 kV to generate electric field strength of 0.6kV/cm. The flow rate of the blending solution was 0.02 ml/min.Poly-aspartic acid was added to silk/PEO solution beforeelectrospinning. The electrospun mats obtained were immersed in 100%methanol for 5 mM to induce an amorphous to β-sheet conformationaltransition, required to preserve the intact membranes in aqueoussolution during the subsequent mineralization steps.

Mineralization

The alternate mineralization process was used to nucleate and growapatite on silk fibers⁵⁷. First, a silk mat (5 mm×1 mm×50 μm) was soakedin 200 mM CaCl₂ solution (buffered with 50 mM Tris·HCl, pH 7.4 at 37°C.) for a predetermined period of time. The silk mat was thentransferred to a 120 mM Na₂HPO₄ solution after excessive moisture wasremoved by blotting on filter paper and soaked for a predeterminedperiod of time. This cycle was repeated depending on the specifics ofthe experiment.

I. Mechanical Properties

The mechanical properties of specimens were measured with an Instron8511 servo-hydraulic-driven tension/compression/cyclic testing machine.All samples were stored in vacuum at room temperature before test. Eachtest was performed 5 times.

II. Characterization

Scanning of some specimens were coated with gold before SEM.

Electron Microscopy (SEM)

Silk fibers and minerals formed were examined using a LEO Gemini 982Field Emission Gun SEM (Thornwood, N.Y.) for morphology analysis.

X-ray Diffraction (XRD)

Silk fibers and minerals were analyzed with a Bruker D8 Discover X-raydiffractometer (Billerica, Mass.) with GADDS multiwire area detector.WAXD (wide angle X-ray diffraction) experiments were performed employingCuKa radiation (40 kV and 20 mA) and 0.5 mm collimator. The distancebetween the detector and the sample was 47 mm.

X-ray Photoelectron Spectroscopy (XPS)

XPS was performed using a PHI 5600 ESCA microscope (Physical Electronics(PHI), Eden Prairie, Minn.). Spectra were typically acquired at apressure below 2×10⁻⁹ Torr using a monochromatized Al Kα sourceoperating at 100 W. Pass energies used for survey and narrow scans were187. 85 and 23.5 eV, respectively. Sample charging was compensated usingfluxes of low energy electrons and positive argon ions.

X-ray Energy-Dispersive Spectroscopy (EDS)

The energy dispersive x-ray spectra were acquired using an EDAX Genesissystem (Mahwah, N.J.) with a sapphire detector and a super ultra thinwindow. The SEM was a FEI Quanta 200 (Peabody, Mass.) tungsten filamentsystem equipped with a EDAX Genesis energy dispersive x-rayspectrometer. The Quanta system was operated in conventional SEM mode.The x-ray spectra were collected for 100 seconds with an acceleratingvoltage of 20 kV, a time constant of 50 microseconds, and a resolutionof 134 eV. The standardless quantification routine was used to determinethe percent of elements present in the spectra.

Results and Discussion

Electrospun Silk/PEO Fibers

Electrospinning is an efficient fabrication process to assemble polymermats composed of fibers with diameters ranging from several microns toless than 100 nm. In this electrostatic process, the polymer solution issubjected to a high-voltage electric field and small fibers are producedupon overcoming surface tension. The morphology and diameter of theelectrospun silk/PEO fibers were examined using SEM. Fine, uniformfibers with diameters of 600±80 nm were obtained. The fiber orientationinside the non-woven mat was random. The addition of the ionicpoly-L-aspartic acid resulted in smaller fiber diameters (350±30 nm)because of the higher elongation forces caused by the higher chargedensity carried by the electrospinning jet.

XPS was used to determine the surface features of the electrospun fibermat (Table 1).

TABLE 1 XPS Results from the electrospun silk fibers surfaces O1s C1sN1s Bindign Bindign Bindign energy Atom energy Atom energy Element (eV)% (eV) % (eV) Atom % N1s/C1s 1 530.9 21.7 284.6 61.1 398.4 15.3 0.25 2530.9 24.8 284.6 61.5 398.4 12.1 0.20 1. silk/PEO fiber(80/20, wt/wt);2. silk/PEO/poly-L-aspartic acid (200 mg poly-L-aspartic acid/g silk)The N1s/C1s ratio of the silk mats without poly-L-aspartic acid was0.25. After blending poly-L-aspartic acid, the N1s/C1s ratio decreasedto 0.20, indicating that more PEO distributed near the fiber surfacewhen the fibers contained poly-L-aspartic acid. FIG. 14 shows the highlyresolved fitted C1s spectra and O1s spectra of the fiber mats with andwithout poly-L-aspartic acid. The analysis of the fitted XPS spectraallows characterization of the chemical environment of differentelements. The carbon signal can be resolved into three differentcontributions: (1) hydrocarbon at about 285.0 eV, (2) ether carbon(O—C—O) from the PEO and amine (C—N) at about 286.6 eV, and (3) amide(N—C═O) from protein at about 288.3 eV. The intensity of ether and aminecomponents is 57.8% of the total C1s intensity with poly-L-aspartic acidblending compared to 47.0% without blending. Two components can befitted to the O1s spectra: (1) amide (N—C═O) at about 531.5 eV and (2)ether (C—O—C) from PEO and C—OH from protein at about 532.8 eV. Theintensity of the component at 532.8 eV increased from 31.2% to 47.8%with poly-L-aspartic acid blending. The increase of ether componentintensity also suggested that more PEO was distributed near the fibersurface with poly-L-aspartic acid blending.

Electrospinning of a polymer solution involves rapid solvent evaporationin the millisecond time scale^(58,59). This time frame may not allow thephase-separated regions to coarsen prior to solidification, resulting ina fine phase morphology. In addition, the elongation effect of theelectrospinning process leads to the orientation of polymer moleculesalong the fiber axis^(60,61). Since PEO is water soluble, removal of thePEO revealed the internal structural of the fibers. A cross-section of asilk/PEO (80/20 wt/wt) blend fiber without poly-L-aspartic acid afterPEO extraction in water for 48 hours reveals holes in the fiberreflective of the prior location of PEO prior to extraction. Therefore,the fibers have a morphology with a PEO phase inter-dispersed within thesilk phase as irregular shapes, or the fiber has a cocontinuousmorphology with PEO inter-dispersed within the silk phase as continuousstrands. The fibers had a cocontinuous morphology when poly-L-asparticacid was included.

III. Apatite Deposition on Fiber Mats

Apatite has been used in orthopedic and dental surgery due to itsbiocompatibility and osteoconductivity. Apatite contributes to theformation of strong bonds in bone as well as to early bone formationaround implants^(62,63). The partial dissolution of apatite causes anincrease in supersaturation level of hydroxyapatite in the immediatemicroenvironment of an implant. Also, apatite can act heterogeneouslyand nucleate additional growth of apatite⁶⁴. Recently, a novel apatiteformation process was developed to generate large amounts of apatite ina relatively short time^(28,57,65). Using this process, coatings ofhydroxyapatite on substrates were attained in hours instead of daysusing soaking in simulated body fluid (SBF) or 1.5×SBF^(34,35).

Scattered (nonaligned) apatite particles were deposited on theelectrospun silk fibroin fibers in the absence of blendedpoly-L-aspartic acid. In contrast, aligned growth of apatite along fiberaxes was obtained when the poly-L-aspartic acid concentration wasincreased to 200 mg/g silk. Randomly oriented apatite particles formedon silk fibers at low concentrations of poly-L-aspartic acid. Therefore,the presence of polyaspartic acid on the silk fibers enhanced control ofthe deposition of apatite, reflecting the increased density andlocalization of the acid groups on the aligned silk templates. Humanbone is composed of inorganic hydroxyapatite nanocrystals (50 nm×25 nm×2nm) integrated with collagen fibers. The crystallographic c axis of thehydroxyapatite is preferentially parallel to the longitudinal directionof the collagen fibers⁶⁶. Noncollageous proteins, such as osteonectinand phosphoproteins, are known to bind to collagen, possibly atparticular sites as hole zones. These hole zones are consideredpotential sites for the specific nucleation and growth of hydroxyapatitecrystals that are organized in bands across collagen fibers⁶⁷.Previously, in vitro aligned growth of hydroxyapatite was obtained byusing reconstituted collagen and self assembled peptide-amphiphilenanofibers as scaffolds⁶⁸⁻⁷⁰. Deposition of apatite on raw silk ordegummed raw silk has been studied^(57,71) and no aligned growth ofhydroxyapatite along silk fiber has been previously reported. In thisstudy, poly-L-aspartic acid, as an analogue of noncollageous proteins,was incorporated into electrospun silk fibers and induced the alignedgrowth of hydroxyapatite along the silk fibroin fiber axes.

The microstructure of the electrospun silk fibers with blendedpoly-L-aspartic acid was considered for the formation of aligned growthof apatite. The silk fibers electrospun from silk/PEO (80/20, wt/wt)displayed microphase separation between the silk fibroin and PEO. Athigh poly-L-aspartic acid concentration, phase separation was moreevident visually prior to electrospinning, since the silk/PEO solutionturned turbid after addition of the poly-L-aspartic acid. Further phaseseparation during the electrospinning process would lead to acocontinuous structure with regular silk and PEO strands oriented alongthe fiber axis. The silk fibers showed grooves along the fiber axesafter soaking in 120 mM CaCl₂ solution for 2 hours, which may bepartially due to the dissolution of PEO phase.

Carboxyl groups enhance apatite deposition on polymersubstrates^(23,24,33). The formation of apatite on negatively chargedsurfaces can initiate from complexation with calcium ions, followed bynucleation and crystal growth. Once soaked in CaCl₂ solution, thecalcium ions can coordinate with the carboxyl groups and complexes suchas —COOCa⁺ and (—COO)₂Ca form. Furthermore, bridges between silkmolecules and poly-L-aspartic acid molecules can form to generate agel-like structure along the silk bands. Ca ions can facilitate thegelation of silk/poly-L-aspartic acid system (unpublished data) as wellas silik fibroin alone⁷². Therefore, the aligned growth of the apatitecan be obtained by heterogeneous nucleation of apatite on the silk-richbands with the coordination of acidic groups from the poly-L-asparticacid. In the case of silk fibers without poly-L-aspartic acid, theamount of apatite deposited was lower. Silk fibroin contains hydrophilicpolar groups, 16.5 mol % hydroxyl and 2.9 mol % carboxyl residues, whichwould facilitate the nucleation of apatite through the formation ofion-ion (between Ca²⁺ and carboxyl group) and ion-polar (between Ca²⁺and hydroxyl and carbonyl groups) interactions.

In a control experiment, poly-L-lysine was incorporated into electrospunsilk fibers and studied for mineralization response in a similar fashionto that described for the polyaspartic acid system. No mineral depositedwhen the electrospun silk mat was soaked in the calcium solution first,and only small amounts of mineral formed when the fibers were soaked inthe phorphorous solution first. The positively charged poly-L-lysinecontaining fiber surface did not facilitate the binding of Ca ions.Although a complex structure might form between phosphate ions andsurface amino groups via electrostatic interaction, there isinsufficient subsequent complexation of Ca²⁺ with the preformedphosphate-amino complex. Negatively charged surfaces (poly-L-asparticacid) had more potential to induce the nucleation of hydroxyapatite,consistent with results of previous studies²³. Another factor that mayaffect the HA nucleation is the high surface area of the electrospunfiber mat. The porous structure of the fiber mat could retain more Ca orphosphate solution, which could be released to the mineralization media.This explanation doesn't exclude the possibility that apatite couldnucleate homogeneously in solution and subsequently deposit on the fibersurface.

Soaking time and cycles on the polyaspartic acid containing fibersaffect mineral deposition. Elemental analysis of the materials aftermineralization was performed with EDS. The main elements of themineralized silk mat were carbon, oxygen, calcium and phosphorus. Smallamounts of sodium and chlorine also were present. The carbon and oxygencould be from both silk and deposited minerals, while calcium andphosphorus could only be from deposited minerals. With the increase ofsoaking time and number of cycles, the amount of deposited mineralsincreased. For example, with 200 mg poly-L-asp/g silk fiber had anitrogen peak attributed to the composition of silk that was notdetected by EDS analysis after 3 cycles of 10-min soaking of the fibers(FIG. 15). XPS analysis of the same sample did not show a N1s peak,which also indicated that silk fibers were fully coated with apatiteafter 3 cycles of 10-minute soaking (data not shown). The Ca/P ratio ofthe hydroxyapatite was 1.67, with the chemical formula Ca₁₀(PO₄)₆(OH)₂.The typical Ca/P ratio of the apatite formed in this study by EDSanalysis (Table 2) was much smaller than the stoichiometric Ca/P valueof hydroxyapatite. In comparison, the Ca/P ratio of hydroxyapatite wasabout 1.61±0.02 by the same technique, which is close to the Ca/P valueof 1.664±0.005 provided by NIST for standard hydroxyapatite.

TABLE 2 Ca/P molar ratio of apatite deposited on silk mats Sample Ca/Pno poly-L-asp, 10 minutes, 3 cycles 1.345 ± 0.02 no poly-L-asp, 10minutes, 3 cycles 1.349 ± 0.01 200 mg poly-L-asp/g silk, 10 min, 3cycles 1.334 ± 0.03 200 mg poly-L-asp/g silk, 10 min, 3 cycles 1.374 ±0.04Some sites for the PO₄ ³⁻ could be substituted by HPO₄ ²⁻ and CO₃ ²⁻.Since CO₃ ²⁻ ions were not included in the alternate soaking process,the substitution with CO₃ ²⁻ should be very low. Theoretically, thesubstitution of one HPO₄ ²⁻ for PO₄ ³⁻ results in a half Ca deficiency,which will result in a Ca/P ratio less than 1.67. To maintain the chargebalance, Na ions can substitute for Ca ions. EDS analysis detectedconsiderable amounts of Na for all the samples, which could be partiallydue to the incorporation of Na ions into the apatite lattice or theamorphous phase.

XRD patterns of the mineralized silk fibers showed that the depositedminerals were hydroxyapatite (FIG. 16). With increased soaking times andnumber of cycles, XRD diffraction peaks became sharper, indicatinghigher crystallinity. Comparing the XRD patterns with the hydroxyapatitestandard, the major peaks (002, 211) match. However, the diffractionreflections appeared broadened with respect to those recorded from thehydroxyapatite standard. The peak broadening indicated poorcrystallinity, which could be caused by disorder in the crystal, such asthe substitution of HPO₄ ²⁻ for PO₄ ³⁻ or Na for ions Ca ions. Theseresults are consistent with the fact that the amorphous calciumphosphate precursor gradually converts into stable hydroxyapatite whenapatite forms in aqueous solution. Poorly crystallized apatite layertends to be covered with non-apatitic highly reactive labile ions of CO₃²⁻, PO₄ ³⁻, HPO₄ ²⁻, which can modulate the adsporption of adhesionligands to facilitate cell attachment⁷³. These properties are notexpected from highly crystalline apatite⁷⁴.

The present work shows that the cooperation of silk template and acidicpeptides can provide the molecular design necessary for controlling thenucleation and growth of apatite. Control of nucleation and growth ofapatite can be accomplished on silk structures with 1D (fiber), 2D(film) and 3D (scaffold) features. By controlling the incorporation ofacidic proteins into these structures, hence controlling the location ofthe nucleation site, silk composites in various forms can be obtained.

IV. Conclusion

Non-woven electrospun silk fibroin fibers were prepared and used asmatrices for controlled apatite deposition to form nanaocomposites.Aligned growth of polycrystalline apatite along the longitudinal axis ofthe fibers was obtained when the fibers were functionalized withpoly-L-aspartic. As an analogue of noncollageous protein in naturalbone, poly-L-aspartic acid rich in carboxylate groups resulted inenhanced control of growth of apatite. The results suggest newdirections in the design and fabrication of controlled molecularcomposites with relevance to bone substitutes because of the excellentbone-binding property of apatite.

All references described herein are incorporated herein by reference.

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We claim:
 1. A matrix comprising an aligned inorganic coating on aprotein template comprising silk fibroin, wherein the protein templatefurther comprises an anionic polymer, wherein the anionic polymerinteracts with the protein template so that a surface is establishedthat promotes nucleation and growth of inorganic crystals, wherein theanionic polymer comprises poly-L-aspartic acid and the aligned inorganiclayer comprises hydroxyapatite, and wherein the weight ratio of thepoly-L-aspartic acid to the silk fibroin is between 200 mg/g and 1 g/g,inclusive.
 2. The matrix of claim 1, wherein the protein templatefurther comprises at least one protein selected from keratins, collagensand silks.
 3. The matrix of claim 1, further comprising at least oneadditive.
 4. A silk fibroin template comprising an aligned inorganiccoating, and at least one therapeutic or biologically active agent,wherein the silk fibroin template comprises an anionic polymer, whereinthe anionic polymer interacts with the silk fibroin template andpromotes more regular nucleation and growth of inorganic crystals,wherein the anionic polymer comprises poly-L-aspartic acid and thealigned inorganic layer comprises hydroxyapatite, and wherein the weightratio of the poly-L-aspartic acid to the silk fibroin is between 200mg/g and 1 g/g, inclusive.
 5. The silk fibroin template of claim 4,wherein at least one therapeutic or biologically active agent isselected from the group consisting of: a small molecule, a protein, anantibody, a peptide, a nucleic acid, a polysaccharide, a glycoprotein,and a lipoprotein.
 6. The silk fibroin template of claim 4, wherein theat least one therapeutic or biologically active agent is a biologicallyactive agent selected from the group consisting of cell attachmentmediators, biologically active ligands, enzymes, and substances thateither promote or inhibit cellular tissue ingrowth.
 7. The silk fibrointemplate of claim 4, wherein the at least one therapeutic orbiologically active agent is a biologically active agent selected fromthe group consisting of collagen, elastin, fibronectin, vitronectin,laminin, proteoglycans, Arg-Gly-Asp (RGD) peptides, bone morphogenicproteins, cytokines, growth factor inhibitors, epidermal growth factor,fibroblast growth factor, platelet-derived growth factor, insulin-likegrowth factor, and transforming growth factor.
 8. The silk fibrointemplate of claim 4, wherein the at least one therapeutic orbiologically active agent is incorporated in, or coated on, the alignedinorganic coating.
 9. The silk fibroin template of claim 4, wherein theat least one therapeutic or biologically active agent is incorporatedin, or coated on, the silk fibroin template.
 10. The silk fibrointemplate of claim 4, wherein the silk fibroin template is selected fromthe group consisting of a silk fiber, a silk thread, a silk mesh, a silkfilm, a silk hydrogel, or a silk foam.
 11. The silk fibroin template ofclaim 4, wherein the silk fibroin template is a three-dimensional poroussilk matrix.
 12. The silk fibroin template of claim 4, furthercomprising a pharmaceutically acceptable carrier.
 13. The silk fibrointemplate of claim 4, wherein the at least one therapeutic agent orbiologically active agent is a therapeutic agent selected from the groupconsisting of anti-infectives, chemotherapeutic agents, anti-rejectionagents, analgesics and analgesic combinations, anti-inflammatory agents,hormones, anti-angiogenic agents, anti-thrombotics, anti-metabolics,antimitotics, anticoagulants, fibrinolytics, growth factor inhibitors,growth factors, vitamins, sedatives, hypnotics, antibiotics, antiviralagents, steroids, growth factor promoters, prostaglandins, andradiopharmaceuticals.
 14. The silk fibroin template of claim 13, whereinthe growth factor is selected from the group consisting of bonemorphogenic proteins, bone morphogenic-like proteins, epidermal growthfactor, fibroblast growth factor, platelet derived growth factor,insulin-like growth factor, transforming growth factors, and vascularendothelial growth factor.